1. Field of the Invention
This invention relates to immobilized enzyme electrodes (hereinafter abbreviated as "enzyme electrode"), and in particular to enzyme electrodes which permit accurate measurements without any influence of electrochemically interfering substances.
2. Description of the Prior Art
Enzyme reactions are widely used in the quantitative analysis of objective substances contained in biosubstances, foodstuffs, etc. because of advantages such as high reaction rate and substrate specificity.
In particular, methods of measuring the concentration of objective substances by use of enzyme electrodes have developed the possibility of repeated use of a minute quantity of enzyme and are spreading their application to the range such as medical service, foodstuff, and chemicals analysis. The amperometric method which measures the current accompanying the electrode reaction of the product by enzyme reaction while applying a constant voltage to the enzyme electrode is widely studied because of the generally simple electrode construction and possible high-sensitivity measurements. Especially, an electrode using an oxidase forming hydrogen peroxide (called H.sub.2 O.sub.2 forming oxidase) reaction system is relatively excellent in response speed and sensitivity. This method, however, has a drawback that the concentration of the objective substance cannot be accurately measured if an oxidizable material is present in the object to be examined.
For example, glucose determination using glucose oxidase (GOD) is described here. ##STR1## The hydrogen peroxide produced according to equation (i) at an enzyme electrode with immobilized GOD causes electrode reaction by a voltage of about +0.6 V to a saturated calomel electrode as shown by equation (ii) EQU H.sub.2 O.sub.2 .fwdarw.O.sub.2 +2H.sup.+ +2e.sup.- . . . (ii)
The current produced here, which is proportional to the glucose concentration, makes the determination of the glucose concentration possible.
The enzyme reaction represented by equation (i) has a high substrate specificity to glucose, the objective substance. However, the electrode reaction represented by (ii) is not specific to hydrogen peroxide and oxidizes various reducing substances, that is, electrochemical interfering substances on the electrode. Thus, the current produced by the oxidation of the interfering substances is added to the measured current, causing an error. It is known that foodstuffs and biological liquid used for measurement contain electrochemical interfering substances such as ascorbic acid, uric acid, reduced glutathione, and tyrosine. Thus, the development of a method for solving the above measuring error has been strongly requested.
A method has been known which answers such a request, in which the current is measured under condition that only hydrogen peroxide is permeated without interfering substances having larger molecular weight than the hydrogen peroxide permeated, by provision of selectively permeable membrane consisting of acetylcellulose, between the immobilized enzyme membrane and the electrically conductive substrate such as platinum in the enzyme electrode for measuring hydrogen peroxide which is produced by the oxidase reaction represented by equation (i).
However, a conventional selectively permeable membrane such as acetylcellulose membrane requires complex adjustment of the degree of acetylation, dissolving method, membrane forming method, and mixing quantity of trifunctional amine, so it is difficult to prepare a selectively permeable membrane of the same quality with good reproducibility.